The development of extremely large male genitalia under spatial limitation.

Extensive research in evolutionary biology has focused on the exaggeration of sexual traits; however, the developmental basis of exaggerated sexual traits has only been determined in a few cases. The evolution of exaggerated sexual traits may involve the relaxation of constraints or developmental processes mitigating constraints. Ground beetles in the subgenus Ohomopterus (genus Carabus) have species-specific genitalia that show coevolutionary divergence between the sexes. Here, we examined the morphogenesis of the remarkably enlarged male and female genitalia of Carabus uenoi by X-ray microcomputed tomography. The morphogenetic processes generating the male and female genitalia at the pupal stage were qualitatively similar to those in closely related species with standard genital sizes. Higher growth rates contributed to the exaggeration of both the male and female genital parts of C. uenoi, possibly related to a gene network commonly upregulated in both sexes. Additionally, the length of the copulatory piece (CP), the enlarged male genital part stored in the aedeagus (AD), reached close to that of the AD at the later developmental stages and thereafter decelerated to grow in parallel with the AD, suggesting a structural constraint on the CP by the outer AD. Then, unlike related species, the lengths of the CP and AD increased at eclosion, suggesting a mechanism leading to further elongation of the male genitalia. These observations suggest that a developmental process allows continuous growth of the male genitalia even under the spatial limitation. These results revealed the spatio-temporal dynamics of the development of exaggerated genital structures under structural constraints.

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles.

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.

Reproductive Character Displacement in Genital Morphology in Ohomopterus Ground Beetles.

AbstractGenital morphology reveals rapid diversification among species, and species-specific divergence in genital morphology may result in reproductive isolation and promote speciation. Natural selection against maladaptive hybridization may cause species-specific genital divergence. In this context, divergence in mating traits is expected to be greater between sympatric populations than between allopatric populations in a pair of species, known as reproductive character displacement (RCD). However, there are few examples of RCD in the genital morphology of closely related species. Additionally, processes leading to RCD have rarely been inferred. In this study, we examined RCD and its underlying mechanisms by focusing on species-specific genital morphologies of closely related Ohomopterus ground beetle species. A morphological analysis showed patterns of RCD in species-specific genital parts in both sexes. Interspecific hybridization was confirmed by a mate choice experiment and by a population genetic analysis indicating extensive interspecific gene flow, suggesting that reinforcement is the most plausible process underlying the observed RCD. We found variation in the degree of displacement in contact zones, which may correspond with the ongoing process of genital evolution and speciation. Our results provide support for the lock-and-key hypothesis of genital evolution in closely related Ohomopterus species.

Global dispersal and diversification in ground beetles of the subfamily Carabinae.

The origin and diversification process of lineages of organisms that are currently widely distributed among continents is an interesting subject for exploring the evolutionary history of global species diversity. Ground beetles of the subfamily Carabinae are flightless except for one lineage, but nevertheless occur on all continents except Antarctica. Here, we used sequence data from ultraconserved elements to reconstruct the phylogeny, divergence time, biogeographical history, ancestral state of hind wings and changes in the speciation rate of Carabinae. Our results show that Carabinae originated in the Americas and diversified into four tribes during the period from the late Jurassic to the late Cretaceous, with two in South America (Celoglossini) and Australasia (Pamborini) and two in Laurasia (Cychrini and Carabini). The ancestral Carabinae were inferred to be winged; three of four tribes (Cychrini, Ceglossini and Pamborini) have completely lost their hind wings and flight capability. The remaining tribe, Carabini, diverged into the subtribes Carabina (wingless) and Calosomina (winged) in the Oligocene. Carabina originated in Europe, spread over Eurasia and diversified into approximately 1000 species, accounting for around 60% of all Carabinae species. Calosomina that were flight-capable dispersed from North America or Eurasia to South America, Australia, and Africa, and then flightless lineages evolved on oceanic islands and continental highlands. The speciation rate increased in the Cychrini and Carabini clades in Eurasia. Within Carabini, the speciation rate was higher for wingless than winged states. Our study showed that the global distribution of Carabinae resulted from ancient dispersal before the breakup of Gondwana and more recent dispersal through flight around the world. These patterns consequently illustrate the causal relationships of geographical history, evolution of flightlessness, and the global distribution and species diversity of Carabinae.

Temporal Variation Dominates in Local Carabid Beetle Communities in Korean Mountains.

Spatial and temporal variation in ecological environments may result in spatial and temporal variation in communities. Temporal studies of biodiversity are essential for forecasting future changes in community structure and ecosystem function. Therefore, determining the mechanisms that drive temporal change in communities remains an important and interesting challenge in ecology. We quantified spatial and temporal variations in carabid beetle communities and site-specific environmental factors for 5 years at nine study sites on three mountains in the Baekdudaegan Mountain Range, Korea. Carabid beetle communities exhibited significant temporal variation, which was larger than spatial variations between and within mountains. Environmental factors mostly varied between sites within mountains. Community variation was only weakly associated with environmental factors at wide scales, i.e., between sites on three mountains, but was strongly associated at narrow spatial scales, i.e., between sites within one mountain. Our results indicate that temporal variation in communities occurs in response to variations in the local climate, and that the patterns of temporal variation differ between mountains. Thus, temporal surveys of insect communities and climates at local scales are important for predicting temporal changes in the communities.

Mesozoic origin and 'out-of-India' radiation of ricefishes (Adrianichthyidae).

The Indian subcontinent has an origin geologically different from Eurasia, but many terrestrial animal and plant species on it have congeneric or sister species in other parts of Asia, especially in the Southeast. This faunal and floral similarity between India and Southeast Asia is explained by either of the two biogeographic scenarios, 'into-India' or 'out-of-India'. Phylogenies based on complete mitochondrial genomes and five nuclear genes were undertaken for ricefishes (Adrianichthyidae) to examine which of these two biogeographic scenarios fits better. We found that Oryzias setnai, the only adrianichthyid distributed in and endemic to the Western Ghats, a mountain range running parallel to the western coast of the Indian subcontinent, is sister to all other adrianichthyids from eastern India and Southeast-East Asia. Divergence time estimates and ancestral area reconstructions reveal that this western Indian species diverged in the late Mesozoic during the northward drift of the Indian subcontinent. These findings indicate that adrianichthyids dispersed eastward 'out-of-India' after the collision of the Indian subcontinent with Eurasia, and subsequently diversified in Southeast-East Asia. A review of geographic distributions of 'out-of-India' taxa reveals that they may have largely fuelled or modified the biodiversity of Eurasia.

Heterochrony and growth rate variation mediate the development of divergent genital morphologies in closely related Ohomopterus ground beetles.

The rapid divergence of genital morphology is well studied in the context of sexual selection and speciation; however, little is known about the developmental mechanisms underlying divergence in genitalia. Ground beetles in the subgenus Ohomopterus genus Carabus have species-specific genitalia that show coevolutionary divergence between the sexes. In this study, using X-ray microcomputed tomography, we examined the morphogenesis of male and female genitalia in two closely related Ohomopterus species with divergent genital morphologies. The morphogenetic processes generating the male and female genitalia at the pupal stage were qualitatively similar in the two species. The male aedeagus and internal sac and female bursa copulatrix were partially formed at pupation and developed gradually thereafter. The species-specific genital parts, male copulatory piece, and female vaginal appendix differed in the timing and rate of development. The relatively long copulatory piece of Carabus maiyasanus began to develop earlier, but subsequent rates of growth were similar in the two species. The timing of the formation of the vaginal appendix and initial growth rates were similar, but subsequent rapid growth led to a longer vaginal appendix in C. maiyasanus. Thus, substantial interspecific differences in the size of genital parts were mediated by different underlying developmental mechanisms between the sexes (i.e., a shift in the developmental schedule in males and a change in growth rate in females). These results revealed the spatio-temporal dynamics of species-specific genital structure development, providing a novel platform for evo-devo studies of the diversification of genital morphologies.

Functional diversity and trade-offs in divergent antipredator morphologies in herbivorous insects.

Predator-prey interactions may be responsible for enormous morphological diversity in prey species. We performed predation experiments with morphological manipulations (ablation) to investigate the defensive function of dorsal spines and explanate margins in Cassidinae leaf beetles against three types of predators: assassin bugs (stinger), crab spiders (biter), and tree frogs (swallower). There was mixed support for the importance of primary defense mechanisms (i.e., preventing detection or identification). Intact spined prey possessing dorsal spines were more likely to be attacked by assassin bugs and tree frogs, while intact armored prey possessing explanate margins were likely to avoid attack by assassin bugs. In support of the secondary defense mechanisms (i.e., preventing subjugation), dorsal spines had a significant physical defensive function against tree frogs, and explanate margins protected against assassin bugs and crab spiders. Our results suggest a trade-off between primary and secondary defenses. Dorsal spines improved the secondary defense but weakened the primary defense against tree frogs. We also detected a trade-off in which dorsal spines and explanate margins improved secondary defenses against mutually exclusive predator types. Adaptation to different predatory regimes and functional trade-offs may mediate the diversification of external morphological defenses in Cassidinae leaf beetles.

Genetic basis of species-specific genitalia reveals role in species diversification.

The diversity of genital morphology among closely related animals with internal fertilization is well known, but the genetic backgrounds are unclear. Here, we show that, in Carabus (Ohomopterus) beetles showing correlated evolution of male and female genital parts, only a few major quantitative trait loci (QTLs) determine differences in genital dimensions between sister species, and sequence divergence is pronounced in the genomic regions containing genital QTLs. The major QTLs for male and female genital dimensions reside in different locations within the same linkage group, implying that coevolution between the sexes is only loosely constrained and can respond to sexually antagonistic selection. The same genomic regions containing the major QTLs show elevated divergence between three pairs of parapatric species with marked differences in genital parts. Our study demonstrates that species diversification can follow coevolution of genitalia between the sexes, even without tight linkage of loci affecting male and female genital dimensions.

Latitudinal variation and coevolutionary diversification of sexually dimorphic traits in the false blister beetle Oedemera sexualis.

Sexual traits are subject to evolutionary forces that maximize reproductive benefits and minimize survival costs, both of which can depend on environmental conditions. Latitude explains substantial variation in environmental conditions. However, little is known about the relationship between sexual trait variation and latitude, although body size often correlates with latitude. We examined latitudinal variation in male and female sexual traits in 22 populations of the false blister beetle Oedemera sexualis in the Japanese Archipelago. Males possess massive hind legs that function as a female-grasping apparatus, while females possess slender hind legs that are used to dislodge mounting males. Morphometric analyses revealed that male and female body size (elytron length), length and width of the hind femur and tibia, and allometric slopes of these four hind leg dimensions differed significantly among populations. Of these, three traits showed latitudinal variation, namely, male hind femur was stouter; female hind tibia was slenderer, and female body was smaller at lower latitudes than at higher latitudes. Hind leg sizes and shapes, as measured by principal component analysis of these four hind leg dimensions in each sex, covaried significantly between sexes, suggesting coevolutionary diversification in sexual traits. Covariation between sexes was weaker when variation in these traits with latitude was removed. These results suggest that coevolutionary diversification between male and female sexual traits is mediated by environmental conditions that vary with latitude.

Impact of sexually antagonistic genital morphologies on female reproduction and wild population demography.

Sexual conflict is a strong driver of evolution. The evolutionary outcomes of sexual conflict can, in turn, influence ecological processes within populations, for example, demography. However, evidence for the latter hypothesis is scarce, especially in the wild. Here, we show that sexual conflict is associated with demographic processes determining population size in the ground beetle Carabus insulicola with elaborate male and female genitalia based on individual- and population-level analyses. We found that sexually antagonistic selection can operate on the genitalia: longer male genitalia can be beneficial in sperm competition but decrease female reproductive success with increased egg dumping, whereas longer female genitalia are resistant to this male harassment via decreased egg dumping and increased fertilization rate. As expected from sexually antagonistic coevolution due to sexual conflict, we detected coevolutionary divergence between male and female genital sizes among populations. In parallel with decrease in female reproductive success, more harmful males with longer genitalia and less resistant females with shorter genitalia were related to small effective population sizes. Thus, sexual conflict may promote coevolutionary diversification between sexual traits, and this was associated with a demographic process. Our findings provide an insight into sex-driven eco-evolutionary dynamics in the wild.

Correction to: Male mate choice in a sexually cannibalistic species: male escapes from hungry females in the praying mantid Tenodera angustipennis.

[This corrects the article DOI: 10.1007/s10164-017-0506-z.].

Male mate choice in a sexually cannibalistic species: male escapes from hungry females in the praying mantid Tenodera angustipennis.

While competing males and choosy females may be common in animal mating systems, male choice can evolve under certain conditions. Sexual cannibalism is such a condition because of the high mortality risk for males. In mantids, female body condition is associated with male mate preference, with fat females preferred, due to at least two reasons: females in poor nutritional condition are likely to attack and predate males, and fat females can potentially increase the number of offspring. Thus, the risk of cannibalism and female fecundity can influence male mating behavior. In this study, we attempted to separate these factors by using the praying mantid Tenodera angustipennis to examine whether male preference for fat female mantids was based on avoiding sexual cannibalism (cannibalism avoidance hypothesis) or preference for female fecundity (fecundity preference hypothesis). The feeding regimes were experimentally manipulated to discriminate between the effects of female fecundity and female hunger status on male and female mating behaviors. We found that recently starved females more frequently locomoted toward the male, and that male abdominal bending was less intensive and escape was sooner from recently starved females. These female and male behavioral responses to female hunger condition may reveal male avoidance of dangerous females in this mantid.

Ecological differentiation and habitat unsuitability maintaining a ground beetle hybrid zone.

Exogenous selection via interactions between organisms and environments may influence the dynamics of hybrid zones between species in multiple ways. Two major models of a hybrid zone allowed us to hypothesize that environmental conditions influence hybrid zone dynamics in two ways. In the first model, an environmental gradient determines the mosaic distribution at the boundary between ecologically differentiated species (mosaic hybrid zone model). In the second model, a patch of unsuitable habitat traps a hybrid zone between species whose hybrids are unfit (tension zone model). To test these, we examined the environmental factors influencing the spatial structure of a hybrid zone between the ground beetles Carabus maiyasanus and C. iwawakianus using GIS-based quantification of environmental factors and a statistical comparison of species distribution models (SDMs). We determined that both of the hypothetical processes can be important in the hybrid zone. We detected interspecific differences in the environmental factors in presence localities and their relative contribution in SDMs. SDMs were not identical between species even within contact areas, but tended to be similar within the range of each species. These results suggest an association between environments and species, and provide evidence that ecological differentiation between species plays a role in the maintenance of the hybrid zone. Contact areas were characterized by a relatively high temperature, low precipitation, and high topological wetness. Thus, the contact areas were regarded as being located in an unsuitable habitat with a drier climate, where those populations are likely to occur in patches with limited precipitation concentrated. A comparison of spatial scales suggests that exogenous selection via environmental factors may be weaker than endogenous selection via genitalic incompatibility.

Embryonic development of Carabus insulicola (Insecta, Coleoptera, Carabidae) with special reference to external morphology and tangible evidence for the subcoxal theory.

The egg morphology and successive changes in the developing embryos of the carabid ground beetle Carabus insulicola (Carabidae) are described based on light and scanning electron microscopy observations. Newly laid eggs of this species are ellipsoid and measure approximately 6.1 × 2.9 mm, before increasing to 6.6 × 3.4 mm at hatching. The egg period is about 11 days at 23°C. The egg shell is characterized by a thin fragile chorion covering a hard serosal cuticle. The embryo forms on the ventral egg surface, where it develops for the duration of the egg period. During the process of thoracic leg formation, two subcoxal rings, subcoxae-1 and 2, are clearly discernible at the basalmost region of the leg rudiments, and these subcoxae participate in the formation of the larval pleura and sterna. The result thus provides tangible evidence for the subcoxal theory, that is, that thoracic pleura and sterna are derived from subcoxal regions. Despite the complete absence of abdominal appendages in the larvae of this species, two pairs of appendage-like swellings, the medial and lateral ones, temporarily arise in the first eight abdominal segments during the middle of embryonic development. The medial swellings are assumed to be serially homologous with the coxal part of the thoracic leg, and they later flatten out and participate in the formation of the larval pleura (hypopleurites). In the light of the serially homologous relationships among gnathal appendages, thoracic legs, and abdominal appendage-like swellings, we identified the subcoxal regions in both the gnathal and abdominal segments. Although, the lateral swellings soon degenerate and disappear, it is considered that the swellings originate in the abdominal subcoxae-2 and may be homologous to the tracheal gills of larvae of Gyrinidae. Based on the embryological results, new interpretations for the constituent of gnathal appendages are proposed.

Colonization and persistence of urban ant populations as revealed by joint estimation of kinship and population genetic parameters.

The decrease in biodiversity due to increasing urbanization has been well documented, but the processes of colonization and maintenance of wildlife populations in urban areas remain poorly understood. We address this issue using 462 individuals from 10 urban populations of the ant Formica japonica in Kobe City, Japan. We sampled workers regardless of colony identity, genotyped them using 6 microsatellite loci, and estimated allele frequencies and genotypes of reproductive individuals, together with other population genetic parameters, by estimating kinship structure using a likelihood method. Estimated genetic diversity and effective size of populations were not associated with environmental parameters, suggesting that populations are unaffected by urbanization. However, effective population sizes were small, and frequent population bottlenecks were detected. These results suggest that urban F. japonica populations are unstable, and the possibility of frequent extinctions and recolonizations in urban habitats. Populations were moderately differentiated without isolation by distance, suggesting a strong dispersal ability that enables colonization of urban habitats. Dispersal was male biased. Collectively, F. japonica was regarded as an urban adapter, which can colonize urban habitats by virtue of its preference for open ground and high dispersal ability but can persist in urban populations for only a short time, showing a tendency as a temporary urban inhabitant.

Mating behavior and the function of the male genital spine in the ground beetle Carabus clathratus.

The morphologies of male genitalia often appear harmful or aggressive, as if they may inflict physical damage upon females during copulation. Such male genitalia are often thought to function in intra- and intersexual interactions during mating. In the carabid genus Carabus, division Spinulati, males possess a spine (spinula) on the intromittent organ, of which function is unknown. To reveal the function of the spinula, we studied the mating behavior and genital coupling of a Spinulati species, Carabus (Limnocarabus) clathratus. The males positioned the spinula along the inner wall of the vaginal opening throughout copulation. This placement created a small dent and subsequently a melanized patch (wound) on the vaginal wall, but the spinula rarely penetrated the vaginal wall. The spinula did not reach the innermost part of the vagina where the spermatophore is deposited. These results suggest that the spinula is not used for inflicting damage on female genitalia or manipulating spermatophores of rival males. During spermatophore formation, the male partially withdrew the aedeagus, and only the aedeagal tip and endophallus remained within the female. By placing the spinula against the vaginal wall, the male could hold the endophallus within the vaginal chamber in the unstable copulatory posture. Thus, our observations suggest that the spinula primarily functions as an "anchor" to maintain the coupling of the male and female genitalia and thereby ensure insemination.

QTL for the species-specific male and female genital morphologies in Ohomopterus ground beetles.

Animals with internal fertilization often exhibit marked diversification in genital morphology among closely related species. However, our knowledge of the genetic architecture underlying genital evolution is still limited. We constructed genetic linkage maps and analysed quantitative trait loci (QTL) for F(2) hybrids of two closely related species of the carabid beetles Carabus (Ohomopterus) iwawakianus and C. (O.) maiyasanus, which show matching male and female genital shapes within species, but marked differences in genital morphologies between species. The linkage maps comprised both amplified fragment length polymorphism and microsatellite markers. Composite interval mapping to detect QTL for three traits of male copulatory piece (length, width, weight) and two traits for female vaginal appendix (length, width) resulted in the detection of one to five significant QTL for each trait. The QTL explained large proportions of phenotypic variance. Thus, the interspecific difference in the genital morphologies appeared to be determined by relatively small numbers of genes with large genetic effects. QTL of different traits for the same or different sexes co-occurred on five of eight linkage groups with significant QTL; in particular, three QTL for different male and female genital traits occurred almost at the same position. Each of the male genital traits showed uniform signs of additive genetic effects, suggesting that directional selection has led to species-specific morphologies. However, the signs of additive genetic effects in each female genital trait were not uniform, suggesting that coevolution between sexes is not necessarily concerted. This result requires further assessment because the sample size of F(2) females was small.

Resource partitioning or reproductive isolation: the ecological role of body size differences among closely related species in sympatry.

1. Body size differences among coexisting related species are common, but the actual effect of these differences in mitigating interspecific interactions, such as resource competition and reproductive interference, is poorly understood. 2. Local assemblages of the ground beetle genus Carabus (subgenus Ohomopterus) typically consist of two or more species of varying sizes. Through foraging and mating experiments using four Ohomopterus species in parapatry and sympatry, we examined whether interspecific body size differences are effective in partitioning food resources or reducing reproductive interference. 3. Because larval Ohomopterus feed exclusively on earthworms, body size differences may be related to partitioning earthworms of different sizes. However, larvae did not exhibit differences in selectivity or attack success on earthworms of different sizes based on larval body size, indicating little possibility of partitioning food by body size. 4. In contrast, interspecific mating behaviours, such as mate recognition, mounting, and copulation, were hindered when body size differences were large; copulation was frequently accomplished between parapatric species with smaller body size differences. 5. These results suggest that body size differences between species effectively reduce reproductive interference, rather than resource competition. Although body size differences in coexisting closely related species have been considered to function in resource partitioning, they may function primarily in reproductive isolation and thereby facilitate coexistence of species.